RESEARCH

PROPOSAL
A fast GC-MS screening procedure for ketamine and its
metabolites in urine samplesmetabolites in urine sample
 Group
members

Baraa yhya Ali Amine Nurrahman

sharif Rachdia Krisna
 Background:

ketamine was introduced in the 1960s as a dissociative anesthetic(1), ketamine

interacts with the N Methyl-D-aspartate (NMDA) channel(2).
It is the psychedelic effects that ketamine shares with PCP, including dream-like
hallucinations, floating sensations, perceptions of creativity and feeling of arousal(3).
Ketamine is also commonly employed as a veterinary anesthetic.
The abuser frequently uses large street doses in an attempt to yield “near-death”
experiences(6).
And there were reports of increased abuse of ketamine and ketamine-related
deaths

 MATERIALS AND METHODS:

Chemicals
Ketamine, NK, ketamine-d4 (K-d4), and norketamined4 (NK-d4) were purchased
from Cerilliant, DHNK was purchased from Formosa Laboratories. Methanol and
ethyl acetate were purchased from Mallinckrodt. Dichloromethane and isobutanol
were purchased from J. T. Baker, USA. Sodium hydroxide, potassium hydroxide,
and concentrated hydrochloric acid were purchased.

All the organic solvents and chemicals are reagent grade.

 MATERIALS AND METHODS:

Urine Samples
Urine samples were collected from participants suspected of abusing ketamine in a
disco-dancing club in Taipei City, Taiwan.

MATERIALS AND METHODS:

Instrument
A Hewlett Packard GC (6890) coupled to a mass detector (5973) equipped with an
autosampler and a HP, 5MS capillary column (12.5 m × 0.20 mm i.d., 0.33 µm film
thickness) (Agilent Technologies, Palo Alto, CA) was used for GC-MS analysis

 PROCEDURE
GC-MS Screening Procedure
1. 1 mL of urine sample was added and spiked with internal standard K- d4 (100 ng in 20 µL of
methanol).
2. The mixture was alkalinized with 1 mL of 1N sodium hydroxide and extracted with 0.2 mL of
hexane by vortex, then stood for 2 min. Eighty microliter of hexane layer was collected for GC-
MS analysis.
3. Two microliter was injected (splitless). Injection port and interface temperature were
maintained at 260˚C and 280˚C, respectively.
4. then held at the temperature for 0.01 min. Flow rate was 1 mL/min. Selected ion monitoring
mode was used for the analysis.
5. The extracted ion ratios were used for quantification. Total analysis time was 5 min per sample.
There was no solvent blank in between samples.
 PROCEDURE
GC-MS Confirmation Procedure

1. urine samples were spiked with 200 ng of K-d4 and NK- d4 as internal standards.
2. Samples extracted with 4 mL of dichloromethane containing 10% isobutanol.
3. The organic layer evaporated to dryness and reconstituted in 100 µL of EA, and 1-3 µL
injected in splitless mode.
4. Selected ion monitoring mode was used for GC/MS analysis.
5. Underlined mass fragments were employed for quantification. Total analysis time was 20
min per sample. A solvent blank was run in between samples

A
I. Performance Characteristics of the
GC/MS Screening

GC-MS chromatogram:
It takes 5.0 min for each sample , including the time required for sample injection
and for the oven to return to initial temperature.
Total ion chromatogram and the extract ion chromatogram of K-d 4 , K, NK, and
DHNK with GC-MS
screening methods were presented in Figures 1 and 2, respectively.

(II) Precision and accuracy:

The within-run precision (% CV) of K, NK, and DHNK at the three
distinct control levels (80, 160, and 400 ng/mL) was all under
8.17%.
The error of accuracy ranges between –12.5% and 5.75% for the
controls.(Table 1)

 II. Performance Characteristics of the

GC/MS Confirmation Procedure:

(I) Precision and accuracy:

Different concentrations of K, NK, and DHNK (0, 100, 200, and 500 ng/mL) were used to
create calibration curves.
Linear regression of the calibration curve for DHNK was y = 0.0019x – 0.0108, R 2 = 0.9984
Three distinct levels of DHNK at 80, 160, and 400 ng/mL have within-run and between-day
precision (%CV) and accuracy (error,%) that range between -9.25% and 9.31%.

(II) Linearity and detection limit:

LOD was 1 ng/mL for K, 5 ng/mL for NK and 20ng/mL for
DHNK
LOQ was 5 ng/mL for K, 10 ng/mL for NK and 40 ng/mL for
DHNK
Range of linearity was 7μg/mL, 4 μg/mL, and 5 μg/mL for K,
NK , and DHNK respectively

(III). Sensitivity and Specificity:

1. 163 urine samples taken from members of disco-dancing clubs were
tested using the GC-MS screening technique.(Results are shown in Table
3)
2. There were 88 positive, 74 negative samples and one false negative
sample found in the results.
3. For K, NK, and DHNK, the screening findings of this one false negative
sample are 98, 20, and 19 ng/mL, respectively. Additionally, the sample's
confirmation findings for K, NK, and DHNK are 103, 37, and 79 ng/mL,
respectively.
4. Sensitivity is found to be 98.9% and specificity is 100%.
Conclusion:
This screening method is fast and sensitive, and it can be applied to
both forensic and clinical toxicological analyses.
 references:

Lin,H.R. and Lua,A.-C.(2005)”A fast GC-MS screening procedure for ketamine and its metabolites
in urine sample”,Journal of Food and Drug Analysis:Vol. 13 : Iss. 2 ,Article 10.
Available at: https://doi.org/10.38212/2224-6614.2538